title: Limitations in small intestinal neuroendocrine tumor therapy by mTor kinase inhibition reflect growth factor-mediated PI3K feedback loop activation via ERK1/2 and AKT.
abstract: BACKGROUND
  Treatment of small intestinal neuroendocrine tumors (SINETs) with mammalian target of rapamycin (mTOR) inhibitors alone or with somatostatin analogs has been proposed as effective therapy, because both agents have been reported to exhibit antiproliferative activity. Because adenocarcinomas escape mTOR inhibition, we examined whether the escape phenomenon occurred in SINETs and whether usage of somatostatin analogs with mTOR inhibitors surmounted loss of inhibition.
  
  
  METHODS
  The effects of the somatostatin analog octreotide (OCT), the mTOR inhibitor RAD001 (RAD), or the combination were evaluated in SINET cell lines (KRJ-I, H-STS) using cell viability assays, western blotting, enzyme-linked immunosorbent assay, and reverse-transcription polymerase chain reaction to assess antiproliferative signaling pathways and feedback regulation.
  
  
  RESULTS
  RAD (10(-9) M) incompletely decreased cell viability (-40% to +15%); growth escape (P < .001) was noted at 72 hours in both cell lines. Phosphorylated (p)mTOR/mTOR and pp70S6K/p70S6K ratios were decreased but were associated with increases in phosphorylated extracellular signal-regulated kinase (pERK)/ERK and pAKT/AKT in both cell lines, whereas phosphorylated insulin-like growth factor 1 receptor (pIGF-1R)/IGF-1R levels were elevated only in H-STS cells. Increased (P < .05) transcript levels for AKT1, MAPK, mTOR, IGF-1R, IGF-1, and TGFβ1 were evident. OCT (10(-6) M) itself had no significant effect on growth signaling in either cell line. An antiproliferative effect (66 ± 5%) using OCT+RAD was only noted in the KRJ-I cells (P < .05).
  
  
  CONCLUSIONS
  SINET treatment with the mTOR inhibitor RAD had no antiproliferative effect based on activation of pAKT and pERK1/2. A combinatorial approach using OCT and RAD failed to overcome this escape phenomenon. However, differences in RAD response rates in individual NET cell lines suggested that pretreatment identification of different tumor sensitivity to mTOR inhibitors could provide the basis for individualized treatment.
authors.affiliation: Gastrointestinal Pathobiology Research Group, Yale University School of Medicine, New Haven, Connecticut 06520-8062, USA.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Svejda
authors.forename: Mark
authors.initials: M
authors.lastname: Kidd
authors.forename: Alexander
authors.initials: A
authors.lastname: Kazberouk
authors.forename: Ben
authors.initials: B
authors.lastname: Lawrence
authors.forename: Roswitha
authors.initials: R
authors.lastname: Pfragner
authors.forename: Irvin M
authors.initials: IM
authors.lastname: Modlin
mesh_terms: D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D000068338:Everolimus; D006801:Humans; D007414:Intestinal Neoplasms / Q000188:drug therapy* / Q000378:metabolism; D007421:Intestine, Small; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D018358:Neuroendocrine Tumors / Q000188:drug therapy* / Q000378:metabolism; D015282:Octreotide / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D020123:Sirolimus / Q000031:analogs & derivatives / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D000068338:Everolimus; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D015282:Octreotide; D019869:Phosphatidylinositol 3-Kinases; D047428:Protein Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D020123:Sirolimus; D058570:TOR Serine-Threonine Kinases; C546842:MTOR protein, human; C535150:MAPK1 protein, human